In tissue engineering, cell biology, and the biomedical field at large, three-dimensional (3D) cell culture provides a tool to more accurately simulate the native in vivo environment for preclinical studies such as drug screening and cellular assays. Recent advances in 3D printing and fabrication technologies have, in turn, advanced the development of these 3D in vitro models. Spheroids, a staple of in vitro 3D culture, have long been employed in the formation and growth of embryoid bodies in embryogenesis, cell clustering for adult tissue growth and organogenesis, as well as cancer and liver organoid toxicity screening.
The ability to reproducibly generate multi-cellular spheroids is essential to provide an effective model of in vivo behavior. The hanging-drop method is a commercially available technique that has been extensively utilized in spheroid culture. This process is labor-intensive due to the need for spheroid transfer and sometimes lacks reproducibility. Micromolding and photolithography have been used to create microwells made of PDMS (polydimethylsiloxane), poly(ethylene glycol) (PEG), or agarose, however, these protocols frequently utilize harsh fabrication processes and produce microwells with limited optical transparency (requiring spheroid transfer for monitoring and imaging), generate multiple spheroids in the same well (in the case of flat wells), or lack control over spheroid placement within the well (making it difficult for high throughput imaging). One example of micro-molded wells for cell clustering is disclosed by Kugelmeier et al. in U.S. Pat. No. 8,911,690. The described well plates, which are available commercially under the name AggreWell™ (STEMCELL Technologies), are produced by one or more known mechanical and chemical processing techniques, such as molding, high-speed cutting, laser cutting, etching, etc. “Customization” of the sizes of the cavities for special applications is achieved through the use of filling inserts or dividers that are positioned when the wells are seeded. For practical reasons, these inserts typically have vertical sidewalls and, thus, modify the wall angles, making them less optimal for uniform cell aggregation.
While 3D spheroid culture are excellent tools for patterning and upscaling stem cell aggregates in a highly controlled manner, the dynamic changes in 3D shapes of the pluripotent stem cell (PSC) aggregates raise greater issues in understanding and controlling early embryogenesis stages. During early stages of embryogenesis in vivo, the PSC aggregates lose their circular symmetrical shapes and transform into polar and non-spherical structures, which then lead to the emergence of multiple different germ layers at separate regions. Such germ layer commitment is believed to be highly related to the 3D shapes of the PSC aggregates in addition to chemokine signaling. The ability to controlling the 3D PSC aggregate shapes in vitro would a powerful way to study the independent effects of shape and polarity on PSC lineage commitment.
Ideally, for cell aggregate culture, especially for screening purposes, the underlying material should consist of an optically clear substrate that encourages single 3D structure growth in the middle of the well, without the need for transfer to another plate, and limits protein deposition that could affect cell-cell attachment and spreading.